Seed Vigor Tests
In 1876, Nobbe in his "Handbook der Samenkunde" described his observations regarding the differences in seed vigor and "germination energy". Since that time, the nature and importance of seed vigor has increased steadily.
Today, the Vigor Test Committee of the Association of Official Seed Analysts has adopted the following definition of seed vigor: "Seed vigor comprises those properties which determine the potential for rapid uniform emergence and development of normal seedlings under a wide range of field conditions." Seed Vigor Testing Handbook Prepared by the Seed Vigor Testing Committee of the Association of Official Seed Analysts (1983).
Biologically, seed vigor is based on the genetic constitution of seeds which establishes their maximum physiological potential based on the fact that seeds begin to deteriorate at maturity and this deterioration proceeds until all of the seed tissues are dead. Id. The rate of deterioration, including loss of vigor, is determined not only by heredity, but also by events occurring during seed development, harvesting, conditioning, and storage. Id.
Several categories of seed vigor tests are known. These categories are: (1) seedling growth and evaluation tests (which are often referred to as "seedling vigor classification and seedling growth rate" tests); (2) stress tests; and (3) biochemical tests.
Germination Testing
In addition to vigor testing, germination testing is frequently conducted to determine seed quality. Germination (in laboratory practice) is defined by the AOSA as, "the emergence and development from the seed embryo of those essential structures, which, for the kinds of seed in question, are indicative of the ability to produce a normal plant under favorable conditions." Id. Germination test results establish the maximum plant producing potential of seed lots and correlate quite well with emergence under favorable field conditions. Id.
Today, the germination test is the principal and accepted criterion for determining seed viability. The test results are typically obtained from seeds which have been placed under favorable germination conditions. Essentially, germination tests are made on artificial, standardized, essentially sterile media, in humidified, temperature controlled germinators for periods sufficiently long to permit rather "weak" seeds to germinate. Id. However, one problem with that prior art germination test is that it overestimates field emergence because rarely, if ever, are favorable conditions encountered in the field.
Another problem with the prior art germination test is that it is scaleless or dimensionless. Either a seed germinates or it does not. Thus, every germinable seed is by definition equal in plant producing ability. Id. The results of a germination test are expressed as a percentage from 1-100%. The problem with using such percentages is that it is very misleading. For example, a seed lot germinating 50% should produce the same stand as a seed lot germinating 100%, provided twice as many seeds are planted. Id. In a few situations it might, but mostly it will not.
Types of Vigor Tests
a. Seedling growth and evaluating tests
Some vigor tests are conducted under the same conditions as the standard germination test, except seedling growth is measured or evaluated in a different way. Seedling growth and evaluation tests are generally inexpensive and relatively rapid. However, the drawbacks of these tests are that conditions are tough to standardize between laboratories and the seed analyst must be able to determine whether the seed has germinated.
The seedling vigor classification is similar to the standard germination test. The only difference between the two tests is that normal seedlings are further classified as "strong" or "weak". Id. A seedling is often characterized as weak if it is missing its primary root and/or cotyledon, if its hypocotyl has breaks, lesions, necrosis, twisting or curling. Id. In contrast, normal seedlings are characterized as "strong". Based on this test, seedlings are divided into those with deficiencies and those without deficiencies. While this test involves very little work, careless handling of the test can result in errors.
The seedling growth rate test involves a measurement of seedling growth. Under this test, seeds are germinated according to the standard germination test with a more specific moisture content of paper towels. Id. At the end of the germination period, seedling growth is measured. Id. Usually, linear growth and dry weight are determined. Seeds which produce a single straight shoot or root can be measured to determine linear growth. The seedling growth rate test suffers from four limitations: (1) the seedling measurement and the removal of cotyledons or other storage tissues prior to oven drying are relatively time consuming; (2) seedling elongation can be inherently different among cultivars; (3) rate of germination is affected by moisture and temperature; and (4) seed size affects hypocotyl growth in soybeans. Id.
b. Stress tests
Various types of stress tests are known. Some of the stress tests simulate stresses seeds encounter in the field. The theory behind a stress test is that under suboptimum or stressed conditions, high vigor seeds have a greater potential for emergence.
In the accelerated aging test, for example, seeds are placed in temperature of 40.degree.-45.degree. C. and nearly 100% relative humidity for various lengths of time, after which a germination test is conducted. This test is relatively inexpensive.
The cold test simulates early spring field conditions by providing high soil moisture and low soil temperature. Typically, seeds are placed in soil in a plastic box or in paper towels lined with soil and incubated at 10.degree. C. for a specified period. Id. At the end of the cold period, the tests are transferred to a favorable temperature for germination. Id. The emergence percentage is considered as an indication of seed vigor. Id. However, one problem with the cold test is microorganisms. Microorganisms frequently cause seed decay, fungus and other problems. In addition, specific soil conditions are often difficult to standardize from laboratory to laboratory.
The cool germination test involves germinating seeds in darkness at constantly low temperatures, such as 18.degree. C. for several days. Basically, this test is a type of seed exhaustion test. This test is also referred to as the slant board test, which has been used to predict the field vigor in lettuce, carrots, cauliflower seeds and cotton. See O. E. Smith et al., "Studies on Lettuce Seed Quality: I. Effect of Seed Size and Weight on Vigor," J. Amer. Soc. Hort. Sci. 98(b): 529-533 (1973). McCormac, A. C. et al., "Automated Vigour Testing of Field Vegetables Using Image Analysis," Seed Sci. and Technol. 18:103-112 (1990).
c. Biochemical Tests
Biochemical tests measure certain metabolic events in seeds that are associated with germination and can be used to assess vigor.
The tetrazolium test measures dehydrogenase enzyme activity. These enzymes reduce tetrazolium chloride salt, which is colorless, to form a water insoluble red compound, formazon, which "stains" living cells a red color. The dead cells remain colorless. See the Seed Vigor Testing Handbook Prepared by the Seed Test Committee of the Association of Official Seed Analysts (1983).
Conductivity tests involve measuring soak water conductivity. Low vigor seeds often have poor membrane structure and often leak. Seeds with such a poor membrane structure frequently lose electrolytes, such as amino acids and organic acids, when they imbibe water, thereby increasing the conductivity of the soak water.
Image Analysis
Image Analysis, which is also known as Machine Vision, is a computer based system that is being used in the plant industry. The most common components of an image analysis system are a camera, a frame-grabber to digitize the analogue image and store it in RAM, a computer to run image-processing, image analysis classification and user access software, and data output hardware such as a monitor and printer. See Draper, S. R. et al., "Machine Vision for the Characterization and Identification of Cultivars", Plant Varieties and Seeds 2:53-62 (1989). Image analysis provides a new way of studying and analyzing plants and seeds. For example, image analysis is being used to analyze and record the shape of plant organs and seeds. Draper, S. R. et al., "Preliminary Observations with a Computer Based System for Analysis of the Shape of Seeds and Vegetative Structures," J. Nata. Inst. Agric. Bot. 36:387-395 (1984). Travis, A. J. et al., "A Computer Based System for the Recognition of Seed Shape," Seed Sci. & Technol. 13:813-820 (1985). Image analysis is also being used to determine the shape and size of plants in order to help classify, characterize, identify, and register new plant varieties. See Keefe, P. D. et al., "An Automated Machine Vision System for the Morphometry of New Cultivars and Plant Gene Bank Accessions"; Draper, S. R. et al, "Machine Vision for the Characterization and Identification of Cultivars," Plant Varieties and Seeds 2:53-62 (1989).
Image analysis has also been used to help visually select healthy plantlets from an array of growing specimens and effect their transfer to separate growth pots. Once transferred to the separate growth pots, image analysis is used to continue to monitor the plantlets until they develop into salable specimens. See He, W. B., et al., "Processing of Living Plant Images for Automatic Selection and Transfer," Computers and Electronics in Agriculture, 6:107-122 (1991).
Image analysis is also being used in determining leaf anatomy. For example, leaf areas and leaf lengths have been measured using image analysis in leaves dissected from the wildtype Arabidopsis thaliana. See Pyke et al., "Temporal and Spatial Development of the Cells of the Expanding First Leaf of Arabidopsis thaliana (L.) Heynh", J. of Exp. Bot. 42:1407-1416 (1991).
Image analysis is also being used in vigor testing and germination testing. Image analysis has been used to measure the results of the slant board test, the accelerated aging test and the cold test. See Keys, R. D. et al., "Automated Seedling Length Measurement for Germination/Vigor Estimation Using ACASAS (Computerized Automated Seed Analysis System)," J. of Seed Technol. 9:40-53 (1984). McCormac, A. C. et al., "Cauliflower (Brassica oleracea L.) Seed Vigour: Imbibition Effects," J. of Exp. Bot. 41:893-899 (1990); McCormac, A. C. et al., "Automated Vigour Testing of Field Vegetables Using Image Analysis," Seed Sci. & Technol. 18:103-112 (1990).
The problem with the prior art seed vigor tests is that most of these tests are run under specific, controlled growing conditions or use chemicals to assess the vigor of the seedlings. Therefore, special manipulation of the seed must take place in order for the vigor rating of the seedlings to be determined.
In addition, most seed vigor and germination tests must be conducted by Registered Seed Technologists (RST). In order to become an RST, the following requirements, established by the Society of Commercial Seed Technologies, must be met:
1. Accumulate a minimum of 100 points from two or more of the following categories: (category C, D or C/D combination is mandatory). PA1 2. Submit to Board of Examiners at the time of examination a seed collection. (Minimum 150 kinds) PA1 3. Attain passing grades in the prescribed examination.
A. Accepted accredited courses in Botanical Science of Seed Technology--2 points for each earned quarter credit hour, 3 points for each earned semester credit hour. Maximum of 50 points allowed. PA2 B. Accepted seed schools--10 points for each week of verified attendance. Maximum of 20 points. PA2 An additional 5 points will be allowed in this category for full attendance at an AOSA-SCST Annual Conference. (Prior to taking the examination.) PA2 C. Training under direct supervision of a qualified Seed Technologist with approximately equal time in purity analysis and germination. 1 point for each 80 hours training. A minimum of 50 points required. PA2 D. Unsupervised testing experience in purity and germination under the guidance of a qualified tutor. 1 point for each 160 hours experience. Minimum of 25 points required. PA2 E. Combination of C and D with one (1) year minimum and 25 and 12.5 points minimum respectively in each category. PA2 F. Attendance at a Seed Testing Workshop. (Maximum of 10 points for verified attendance)
Constitution and By-laws of the Society of Commercial Seed Technologies (1992)
The United States Department of Agriculture (USDA) supports and promotes this program. For example, in order for seed to be certified for export, an RST must have conducted the germination test.
The vigor rating of this invention does not require that the seeds be grown under specific, controlled growing conditions. Under this invention, the vigor rating can be determined on seedlings grown under any type of growing conditions. For example, the vigor rating of seedlings grown under optimum, greenhouse or outdoor conditions can be determined.
In addition, under the seedling growth rate test, every factor of growth (root size, leaf size, stem size, etc.) is measured as part of the test. The concept is that the lot that grows the most from day-to-day is the most vigorous. Under the present invention, the vigor rating is determined by testing the seedling at any stage of development as long as the leaves are present and they do not overlap.
Additionally, the prior art seed vigor and germination tests have required an RST to conduct the vigor testing. Thereupon, a grower growing a plug flat of trays has to locate an RST to determine the vigor rating of his seedlings. With the present invention, an RST is not needed. Instead, anyone can be trained to conduct the vigor testing of this invention.
Therefore, the present invention provides a means be which a grower can determine the vigor rating of a plug flat of seedlings grown under any kind of growing conditions and at any stage of development.